INDUCTION OF ESTRUS, OVULATION AND

FERTILITY IN PREPUBERAL GILTS

R. D. BAKER, B. R. DOWNEY

To cite this version:

R. D. BAKER, B. R. DOWNEY. INDUCTION OF ESTRUS, OVULATION AND FERTILITY
IN PREPUBERAL GILTS. Annales de biologie animale, biochimie, biophysique, 1975, 15 (2),
pp.375-382. <hal-00896965>

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INDUCTION OF ESTRUS,
OVULATION AND FERTILITY IN PREPUBERAL GILTS
R. D. BAKER

B. R. DOWNEY

NI acdonald Embryo Unit,

,1Iacdonald College of hlcGill University,
Ste A
nne de ]3ellevzie, Ouebec (Canada) HOA ICO

SUMMARY
Induction of synchronized estrus and ovulation in prepuberal gilts is utilized in our laboto study factors influencing fertility and early embryonic development. Prepuberal females
provide an inexpensive source of fertilized eggs, and they are not complicated by the presence of
estrous cycles. Thus, estrus inducing treatments can be applied at any time.
In the first of four experiments reported here, a single injection of 5 mg gonadotrophin releasing hormone (GnRH) was not effective in causing either estrus or ovulation. In the second study,
a single injection of either 5 or io mg GnRH in combination with 300 IU
pregnant mares serum
gonadotrophin (PMSG) resulted in ovulation in less than 50 p. 100 of gilts treated. The dose of
GnRH had no influence on the number of females ovulating or on ovulation rate.
When gilts were given an injection of 200
, 300
, 400 or 500 IU PMSG in combination with
either 200 or 300 IU human chorionic gonadotrophin (HCG), ovulation occurred in 95 p. 100 of
0 animals treated. Laparotomies in 1
8
6 females (
2 per group) indicated that most ovulations took
place between I Io and 120 hours after the injection regardless of the dose. All gilts were slaughtered seven days after the injection and three days after insemination. Fewer (P < o.oi) ovulations were observed in gilts injected with 200 IU PMSG than with other doses of PMSG and
fewer (P < )
05 fertilized eggs were recovered from gilts injected with 200 IU than with either
.
0
300 or 400 IU PMSG. Ovulation rate (P < o.o!) and ovarian weight (P < o.ol) were increased,
but uterine weight (P < o.or) was reduced when the dose of HCG was increased from 200 to
300 IU. Mean maximal number of fertilized eggs (
20 per gilt) was recovered from females injected
with 400 IiJ PMSG plus 300 IU HCG.
In the final experiment, implants or single injections (
40 mg) of ethinyl estradiol resulted in
estrus and ovulation in a majority of the females treated, but fertility was not assessed. Further
research is needed to define mechanisms and to determine resultant pregnancy rates in estrogen
and gonadotrophin treated prepuberal gilts.

ratory

INTRODUCTION

As

early

as

, Casida demonstrated that ovulation could be induced in

1935

prepuberal gilts by giving multiple injections of pregnant mares serum gonadotrophin (PMSG) or purified pituitary powder. These observations were confirmed by

54 and DzmK and G!H!,sACH (

g
I
(
)
68, BAKER and
g66). In 19
I
CoGGINS reported that the number of ovulations increased linearly between 7
2 and
.
as
the
dose
of
was
increased
to
none
of
PMSG
from
2000
IU.
However,
45.8
250
gilts
showed good standing estrus and fertilized eggs were recovered from only 55 p. 100
of the treated gilts. In addition, pregnancy in these gonadotrophin treated, prepuberal gilts was terminated by regression of the corpora lutea at about 2
o days of
Du M
UISSON
ESNIL du B

gestation HA
(S
w
et al., I
I ; SEGAL
7
g
More recently, an injection of

and BAKER,

Ig73).

synthetic gonadotrophin releasing hormone

(GnRH) increased serum FSH and I,H in rats (A
) and increased
RIMURA
et at., 1972
serum I,H in prepuberal pigs (C
R et al., Ig73).
E
Y et al., 1973 and BAK
T
HAKRABOR
In the latter study, ovulation was induced by injecting 1
0 mg GnRH into gilts
.
whose ovaries had been stimulated to form ovulable follicles by injecting PMSG.
The purpose of experiment I in this study was to see if a single injection of 5 mg
of GnRH would result in estrus and ovulation in prepuberal gilts. Recently, I,AMORD!
a

and T
LD
F
N
E
B
STA
E )
1974 reported that an injection of 3 mg GnRH resulted in vulva
(
and
swelling
standing estrus in prepuberal gilts within 24 hours of the injection.
2IUK 4
D
g6 observed ovulation in 75 p. 100 of the prepuberal gilts given a
I
(
)
single injection containing 250 IU PMSG and 250 IU human chorionic gonadotrophin
(HCG). However, this treatment did not result in the onset of normally recurring
estrous cycle. More recently, a combination of 400 IU PMSG and 200 IU HCG
resulted in both synchronous estrus and high fertility in prepuberal gilts (S
CHILLING
and CE
E, 1972
RN
AMA
J
RA
E
H
AN,
N Ig73).
, and BAKER and DR
ooo IU does not usually result in ovulaInjecting PMSG alone in doses of up to I
tion in prepuberal pigs weighing less than 85 kg (BAKER and CoGGINS, 19
66, and
the
addition
of
IU
HCG
to
or
IU
o
20
PMSG
A
B
R
E
et
K c!t., ).
300
400
73 Whereas,
g
I
role
of
is
not
understood.
causes
estrus
and
ovulation.
The
the
HCG
fully
consistently
It could act directly on the ovary to stimulate an increase in endogenous estrogen
production or it could act by increasing the release of endogenous gonadotrophins
or both. Experiment 2 was designed to see if GnRH could be used as a substitute
for HCG in the PMSG/HCG combination.
Experiment 3 was designed to further investigate the influence of doses of
PMSG and HCG in the PMSG/HCG combination on estrus, ovulation and ferlitization.
Since DzIUK 4
g6 reported that an orally active estrogen, ethinyl estradiol
I
(
)
in
and ovulation in prepuberal pigs, the use of estrogens to
resulted
estrus
(EE),
control ovulation in pigs has received little attention. The final experiment was designed to study the potential usefulness of either estrone or EE given as an injection
or

implant.
MATERIALS AND METHODS

The 1
5 animals used in this study were Landrace or Landvace X Yovkshive, Hampshive
6
Du
o
y
c crossbreds. They weighed !j to 95 kg, were i6o to i8o days of age, were housed in pens
of four to eight, were full-fed a i
4 p. IOO protein finishing ration and were randomly allocated to
the treatment groups within each experiment.
The gilts were observed daily for estrus in the presence of a teaser boar. Signs of estrus were
rated as 3 (standing heat), 2
(swollen vulva and almost standing), I (slight swelling of vulva)
or o (no indication of heat). Gilts rated 2 or 3 before the experiment started were considered to
have reached puberty and were excluded from treatment.
or

Experiment1
Twelve gilts were used. Six were injected intramuscularly with 5
,
mg GnRH (AY 24031
Ayerst Laboratories) in 5 ml of saline, and six were injected with5
ml of saline to serve as controls.
the
animals
for
as
described
above.
were observed twice daily
Following treatment,
signs of estrus
Seven days post treatment, all r2 gilts were killed, reproductive tracts were removed and ovaries
were weighed and examined for number and size of follicles and for ovulation points. The uterine
horns were dissected free of ligaments and weighed.

Experiment 2
Thirty-six gilts were assigned to one of four treatment groups : (
) 300 IU PMSG (Equinex
1
,
R
Ayerst Laboratories) plus 200 IU HCG (APLR, Ayerst Laboratories) ; (
)
3
) 300 IU PMSG alone ; (
2
300 IU PDZSG plus 5 mg GnRH ; and (
) 300 IU PMSG plus io mg GnRH. All hormones were
4
administered as a single intramuscular injection. All gilts were checked for estrus as described
above and were artificially inseminated 9
6 hours after the injection. Care was taken to minimize
the stress of insemination in those gilts not in standing estrus. All gilts were inseminated with 8
0
to 100 ml of fresh, undiluted semen from a fertile boar using a rubber corkscrew tipped (Melrose)
pipette. Seven days after injection, the animals were slaughtered and eggs were recovered by
flushing the oviducts and uterine horns with about 20 ml of Tyrodes solution. Ova were mounted
and examined for presence of spermatozoa and stage of development using phase-contrast microscopy.

Experiment 3
Each of
of the

eighty-five gilts were given a single intramuscular injection of PMSG and HCG
following combinations (IU PISG/IU HCG) : 200/200
, 200/3
, 0,
0
00/20 300/300,
3
00/200, 00,
4
0/3 50
0
4
0 and 500/300
20
0/
. At least ten females were used per treatment group.
Estrus was assessed and inseminations were performed as described in experiment 2
. In addition, two gilts in each group were anesthetized (Na pentobarbital followed by halothane) and
laparotomized, mid-ventrally, y to i2o hours following the injection to assess the time of ovulation. Care was taken to minimize the handling of the ovaries and oviducts during surgery. The
gilts were slaughtered seven days following the injection, reproductive tracts were removed and
in

one

ova were

recovered

as

described above.

Experiment4
Each of the 32 gilts was assigned to one of four groups. Treatments included (
) 40 mg of
1
estrone given subcutaneously in 4 ml of corn oil, (
) 40 mg of ethinyl estradiol given subcuta2
neously in corn oil, (
) implants containing 200 mg estrone, and (
3
) implants containing 200 mg
4
R
ethinyl estradiol. The implants were 5
. cm long and were prepared from medical-grade Silastic
tubing (ID 4
7 mm X OD 7
.
9 mm, Dow Corning). The steroid was placed in the lumen of the
.
R Medical Adhesive (Silicone Type A, Dow Corning).
tubing and both ends were closed with Silastic
The implants were inserted subcutaneously under the loose skin in the neck behind the ear.
The gilts were observed for estrus twice daily after treatment but were not inseminated.
All 32 animals were anesthetized and laparotomized mid-ventrally seven days after the injection
or insertion of the implant. Ovaries were examined for follicular development and for approximate time and number of ovulations. Uterine development was noted.

RESULTS AND DISCUSSION

In contrast to a recent report (I,AMOxD! and LD
TAB
S
F
N
E
T,
E 1974
) that a single
of 3 mg GnRH induced standing estrus and ovulation in prepuberal gilts,
none of the pigs in our study that were given 5 mg GnRH showed either standing
estrus or ovulation (table z). Three females did show some vulva swelling about 3
6
to 4
8 hours after the injection, and at slaughter seven days after the injection, two

injection

Annales de

Biologie animale.

.
1975

of three gilts with a swollen vulva and one without any external signs of estrus had
follicles larger than 6 mm. Even though the follicles were unusually large,there was
no indication that they were
going to ovulate.

GnRH

shown to induce ovulation in PMSG-primed proestrus

the time and value of the maximum serum LH
concentration varied greatly. Gilts that ovulated following the GnRH injection
had a mean maximum I,H level of 2
.8 ng/ml which occurred approximately 14 hours
after the injection. The authors are not aware of any published report on serum FSH
concentration in prepuberal gilts following either PMSG or HCG administration.
Such data would undoubtedly help to explain the effects of these hormones.
PMSG is a potent stimulator of follicular development in most mammalian
species studied to date (Na!,snNnov, 195
8). In the prepuberal pig, ovulation does
not normally occur as a result of a single injection of less than 750 IU PMSG (BAKER
et al., 1973
). However, the simultaneous administration of HCG and PMSG
resulted in follicular growth and ovulation when as little as 125 IU of each gonado-

Although

was

prepuberal gilts (B
R
E
AK
et at., 1973
),

trophin

was

given (DzmK, i964).

The role of HCG in causing ovulation when administered with PMSG is not
clearly understood. BAKER and A
AJAMAH
R
N
E
N
DR )
1973 estimated that gilts given
(
a PMSG/HCG combination required approximately 7
8 hours to develop follicles
of
to
an
stimulus.
Since
HCG has a relatively short
capable
ovulatory
responding
half-life in blood (P
and
it
is
WARD, z
unlikely that the HCG is serving as
ARLOW
6t),
9
the ovulatory stimulus. A more likely possibility is that the HCG is stimulating
an increase in estrogen production from the developing follicles and that the resultant endogenous LH release causes ovulation.
If the above hypothesis is true, it was reasoned that GnRH might serve as a
satisfactory substitute for HCG in the PMSG/HCG combinations. Experiment 2
was conducted to test this possibility. Combining either 5 or 10 mg GnRH with
300 IU PMSG had no positive influence on estrus, ovulation or fertility. Therefore,
it was concluded that 5 or 10 mg GnRH, administered with PMSG, does not contribute toward follicular maturation and ovulation as does HCG.

Age of the animals and different batches of PMSG may account for the observation that four of nine gilts receiving only 300 IU PMSG ovulated (table 2
), which
is in contrast with previous reports.

In

, only 24 (
experiment 3
8 p. 100
2
) of the gilts came into standing estrus, although sixty (
71 p. 100
) developed a red and swollen vulva 3
6 to 72 hours following
the injection. None of the gilts were in standing heat earlier than 72 hours after the

nor later than 10

8 hours after the injection. Of animals showing heat, six
in the 300 IU PMSG/
200
300 IU HCG group and five were in the 400 IU PMSG/
IU HCG group. All other groups had three or less gilts showing standing estrus. In
addition, estrus ratings were not significantly correlated with ovulation rate (table 4
),
uterine weight or the percentage of eggs fertilized.

injection
were

>

6
Of the 1

gilts that were laparotomized between

114 and

z2o

hours after

injec-

tion, eight had recently ovulated, four were in the process of ovulating and four had
preovulatory follicles but had not yet begun to ovulate. One of the gilts in the latter
group did not ovulate subsequent to the laparotomy, perhaps because of the surgical
interference. Observations of the ovaries indicated that most of the ovulations occurred
IO and 120 hours after the PMSG/HCG treatment.
between I
At slaughter, data from five gilts in experiment 3 were excluded from the
analysis. Based on the presence of mid-cycle corpora lutea, three females were
judged to have reached puberty before the injection, one gilt had a missing left
uterine horn and the fifth had an infantile genital tract. Two of the three gilts that
had mid-cycle corpora lutea also had more recent corpora lutea (mean of II
) which
likely resulted from the treatment.
The higher dose of HCG in combination with all doses of PMSG resulted in a
greater ovulation rate (P < )
05 and ovarian weight (P < )
.
0
01 but a reduced
.
0
uterine weight (P < ).
01 Since there was no evidence that the gilts ovulated earlier
.
0
following the combinations containing 300 IU HCG, the authors have no explanation for the reduced uterine weight in these gilts.
Five gilts given 500 IU PMSG/
300 IU HCG had more than 35 ovulations.
the
of
However,
percentage
eggs recovered and the percentage of eggs fertilized
were low. In four of the gilts, examination of the ovaries indicated that ovulations
may have occurred over a much wider range of time (up to 4
8 hours) than normal.
an
DzmK
confirms
early report by
Experiment 4
19 that ethinyl estradiol
(
)
4
6
.induces estrus and ovulation in prepuberal gilts. In this study relatively low rates of

ovulation were observed (table 5

). Pigs in Dziuks study were given 20 mg ethinyl
estradiol orally for five days and only four corpora lutea resulted. In both studies,
the gilts had a mean of approximately nine follicles greater than4
mm.

Estrone had no detectable effect on follicular development, estrus or ovulation.

Uterine hypertrophy was noted in six of the gilts that were given implants, but
none of the gilts that were injected.

Colloqtte :

Control

os(,xital cycles iaa 6iojp?estic animals, ,

October 2!-30, 1974. Nott!!iliy.

ACKNOWLEDGEMENTS
The authors arc grateful to George S
HAW and Dou!las .
RVI of Ayerst Research LaboraI
E
B
tories for their advice and for supplying the PMSG, HCG and GnRH. Also, we want to thank
Peter ,
ONLON Barrie Sruwwkl and Ted SUTHERLAND for their assistance in carrying out the
C
study, and Paul LwcuE for his help in preparing the manuscript. The study was supported finan491 from the Ministere de 1Agriculture (in Ouebec and a grant from
72
cially by grant no. -NIcAAverst Research Laboratories.

RSUM
INDUCTION DE

L<RSTRUS

ET DE

CHEZ LES TRUIES

LOVULATION
IMPUBRES

ET

FERTILIT

Bous induisons dans notre laboratoire lestrus synchronise et lovulation chez des truies
afin dtudier les facteurs qui influent sur la fertilit et le dveloppement du jeune
embryon. La truie impubre est non seulement une source conomique dovules fconds mais
elle est aussi libre des complications du cycle &oelig;strien.
insi les traitements pour induire lestrus
A
peuvent tre administrs tous moments.
Dans une premire tude, mg de GnKH donne en injection unique na dclanch ni
lestrus ni lovulation. Dans une seconde tude, aprs une injection unique de ou io mg de

impubres

GnRH combin avec 300 UI de lhormone gonadotrope srique (PMSG), nous avons observ
lovulation dans moins de 50 p. 100 des truies traites. Le nombre de femelles ovulant et le nombre
dovulations ntaient pas fonction de la dose de G
RH. Nous avons cependant induit lovulation
n
chez 95 p. I
OO des 8
0 truies que nous avions traites avec une injection unique de 200
, 300
, 400
,
ou 500 UI de PMSG combin avec 200 ou 300 UI de lhormone
gonadotrope chorionique (HCG).
Lexamen de 1
6 truies (
2 par traitement) par laparotomie nous a permis de dterminer que lovulation avait lieu surtout entre 110 et r2o heures aprs linjection, et ceci indpendemment de la
dose injecte. Toutes les truies ont t abattues sept jours aprs linjection et trois jours aprs
linsmination. Moins (P < o.oi) dovulations ont t comptes et moins (P < )
05 dovules
.
0
fconds ont t recueillis chez les truies traites avec 200 UI de PMSG que chez celles traites avec
300 ou 400 UI de cette hormone. Quand la dose dHCG est passe de 200 300 UI, nous avons
observ un taux dovulation plus lev (P < ),
05 un plus grand poids des ovaires (P < o.oi)
.
0
mais une diminution du poids de lutrus (P < o.oi). La plus haute moyenne dovules collects
fconds a t observe chez les femelles traites avec 400 UI de PMSG et 300 UI dHCG.
Dans la quatrime et dernire tude, un implant ou une injection unique de 4
o mg dthinyl
estradiol a provoqu lestrus et lovulation chez la plupart des femelles traites mais le taux de
fertilit na pas t dtermin. Dautres tudes restent ncessaires pour dfinir les modes daction
et pour quantifier les taux de gestation chez les truies impubres traites avec des estrognes ou
des gonadotropes.

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